Summary A number of new principles have emerged from the study of inorganic physiology, including the idea that intracellular metals such as zinc, copper and iron are not `trace elements' from a cellular point of view. Metallome analysis for many cell types reveals that essential metal ions are routinely maintained in most cells at much higher levels (i.e., 0.6 mM). These insights, as well as the emerging literature linking metal physiology to many disease states, underscore the importance of establishing the fundamental principles, general pathways and macromolecular mechanisms required to manage cellular regulation of millions of metal ions. Our approach to delineating these new principles involves mechanistic and structural characterization of metal receptors that switch on and off genes in a metal dependent manner. This proposal addresses several issues in the field of inorganic physiology. The first question is: how do regulatory metal receptors work within the larger macromolecular complexes that they control, including the multisubunit enzymes RNA polymerase and the ribosome? Our preliminary work indicates that copper and zinc homeostasis in E. coli is under the control of complex transcriptional and translational mechanisms that involve protein-induced distortions in DNA structure. The proposed studies also employ advanced sequencing and quantitative proteomic technology to understand how cells control the overall metal economy. This work is revealing many new metal responsive genes in E. coli, which in turn informs our knowledge of fundamental mechanisms used by pathogens when they are subjected to metal limitation by the host immune system. The specific aims focus on resolving fundamental questions about the structures, functions and molecular mechanisms of these key metal sensing metalloregulatory proteins. The proposed experimental approach will employ x-ray crystallography, biophysical methods, single particle electron microscopy, and proteomic and bioinformatic methods to understand the pathways that E. coli uses to respond to changing metal ion levels in the growth media. The effects of these biophysical switching mechanisms on intracellular metal physiology will then be examined using novel single cell analytical and imaging methods with the overarching goal of establishing general principles and mechanisms that control metal ion homeostasis in normal and disease states.